upstream signal transduction of nf-kb activationatlasgeneticsoncology.org/deep/nfkbid20033.pdf ·...

Atlas of Genetics and Cytogenetics in Oncology and Haematology

Upstream Signal Transduction of NF-kB Activation (Review)

Fei Chen§, Jacquelyn Bower, Laurence M. Demers¶, and Xianglin Shi The Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, WV 26505, USA; ¶Department of Pathology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA § To whom correspondence should be addressed at: Dr. Fei Chen PPRB/NIOSH, 1095 Willowdale Road, Morgantown, WV 26505 Tel: (304) 285-6021, Fax: (304) 285-5938, E-mail: [email protected] December 2001

Abstract

NF-κB is a transcription factor governing the expression of genes involved in the

immune response, embryo or cell lineage development, cell apoptosis, cell cycle

progression, inflammation, and oncogenesis. During past few years, tremendous

attention has been focused on the upstream signaling pathways leading to the

activation of this transcription factor. Many of these signaling molecules can serve as

potential pharmaceutical targets for the specific inhibition of NF-κB activation and the

subsequent interference of disease processes. However, how these molecules

interact with each other is still a debatable issue. Since many nodal signal molecules

in this pathway relay more than one of the upstream signals to their downstream

targets, it has been speculated that the transmission of signals involves a network,

rather than a linear sequence in the activation of NF-κB. Thus, elucidation of the

detailed relationships among the upstream signaling molecules of NF-κB activation

will be important in developing pharmaceutical inhibitors that specifically inhibit the

activation of NF-κB. Such inhibitors would be predicted to have powerful anti-

inflammatory and/or anti- carcinogenic effects.

Atlas Genet Cytogenet Oncol Haematol 2002; 2 -345-

Key words: IKK, kinases, NF-κB, ROIs, signal transduction, ubiquitination.

The NF-κB transcription factor was first discovered in 1986 . Knowledge of the

activation of this transcription factor lagged behind the understanding of its function.

Nevertheless, tremendous progress has been achieved over the last two years

regarding the signal transduction pathways leading to the activation of NF-κB

including the structure and function of IκB kinase complexes (IKK)1, the upstream

signaling pathways, the interactions among diverse signaling components, and the

extracellular regulators .

At present, five mammalian NF-κB family members have been identified and cloned .

These include NF-KB1 NF-κB1 (p50/p105), NF-KB2 NF-κB2 (p52/p100), RelA

RelA(p65), RelBRelB, and c-Relc-Rel . All these NF-κB family members share a

highly conserved Rel homology domain (RHD) responsible for DNA binding,

dimerization, and interaction with IκB, the intracellular inhibitor of NF-κB. The C-

terminal regions of RelA, RelB and c- Rel contain a transactivating domain that is

important for NF-κB-mediated gene transactivation. The C-termini of the precursor

molecules for p50 and p52, p105 and p100, contain multiple copies of the so-called

ankyrin repeat, which is found in IκB family members, including IκBα, IκBβ, IκBε,

Bcl3Bcl3, and Drosophila cactus.

Diverse stimuli, which typically include cytokines, mitogens, environmental and

occupational hazards, toxic metals, intracellular stresses, viral or bacterial products,

and UV light, induce expression of early response genes through the NF-κB family of

transcription factors . In resting cells, NF-κB is sequestered in the cytoplasm in an

inactive form through its association with one of several inhibitory molecules,

including IκBα, IκBβ, IκBε, p105, and p100. Activation of the NF-κB signaling

cascade results in complete degradation of IκB or partial degradation of the C-termini

of p105 and p100 precursors, allowing the translocation of NF-κB to the nucleus,

where it induces transcription (Fig. 1). Activated NF-κB binds to the enhancer or

promoter regions of target genes and regulates transcription of genes mediating cell-

to- cell interaction, intercellular communication, cell recruitment or transmigration,

amplification or spreading of primary pathogenic signals, and initiation or acceleration

of carcinogenesis. The consensus binding site of NF-κB is composed of the

GGGRNNYYCC sequence, where R is purine, Y is pyrimidine, and N is any bases.


Figure 1 : Simplified signal transduction pathways of NF-κB activation. Pro- inflammatory signals,

mainly TNFα, IL-1 or Toll, bind to their corresponding receptors, leading to a recruitment of receptor-

associated proteins, such as MyD88 and IRAK for IL-1R/TLR, TRADD and RIP1 for TNF receptor. In

turn these associated proteins recruit TRAF2 or TRAF6, both of which activate TAK1 possibly through

a non-destructive G76-K63 polyubiquitin chain-dependent mechanism (Ub63). Activated TAK1 or

other MAPKKK family kinases, such as NIK and MEKK1, may phosphorylate and activate IKK

complexes that are responsible for the phosphorylation of the IκB protein. Phosphorylated IκB proteins

are recognized and modified by the G76-K48 polyubiquitin chain (Ub48) via the SCF-β-TrCP complex.

This process is followed by proteasome-mediated degradation of IκBs. Stress signals resulting in the

generation of ROIs contribute to the activation of NF-κB and may involve the sequential activation of

ASK1, SEK1 and JNK. Activated JNK induces the accumulation of β-TrCP protein, which facilitates the

ubiquitination process of IκB proteins. I. IκB kinase complexes (IKK)

The expanding family of IKK, which includes the IKKα, IKKβ, IKKγ, and IKKi/ε, has,

over the past three years, been implicated in the phosphorylation of several IκB

proteins and NF-κB family proteins, such as IκBα, IκBβ, IκBγ, IκBε, p105, p100, and

RelA(p65) in response to numerous and diverse stimuli . In addition, some non-

IκB/NF-κB family proteins, such as β-catenin and HIV vpu protein, have also been

implicated as potential IKK substrates . All IκB proteins contain two conserved serine


(S) residues within their N-terminal domain. Phosphorylation of these conserved S

residues by IκB kinase (IKK) in response to inducers, leads to the immediate

polyubiquitination of IκB proteins by SCF-β-TrCP, a ubiquitin ligase E3 complex .

This modification subsequently targets IκB proteins for rapid degradation by the 26S

proteasome . The first identified IKK complex, which is also the major IKK complex in

most cell types, contains two catalytic subunits, IKKα and IKKβ, and a structural

component named NEMO/IKKγ/IKKAP, which may relay upstream signals to IKK and

possibly promote assembly of the IKK complex .

IKKα and IKKβ share 50% sequence homology. Both proteins contain a N-terminal

kinase domain, a C-terminal region with a leucine zipper, and a helix-loop-helix

domain . An activation loop similar to the one found in the MAP-kinase kinase (MEK)

family of proteins has been identified between the kinase subdomains VII and VIII of

IKKα and IKKβ. Studies by in vitro or ex vivo approaches indicate that both IKKα and

IKKβ are interchangeable in phosphorylating S32/S36 of IκBα, and S19/S23 of IκBβ .

However, substantial differences in function and regulation of IKKα and IKKβ have

been documented. First, IKKβ is far more potent than IKKα in IκBα phosphorylation

in response to proinflammatory stimuli, such as the signals induced by TNFα, IL-1

and LPS . Second, whereas IKKα seems to be more responsible for NF- κB-inducing

kinase (NIK) signals, IKKβ appears more important in mediating MEKK1 reactions .

Third, gene knockout studies demonstrated that IKKα, but not IKKβ, is physiologically

involved in NIK-mediated carboxyl terminal phosphorylation and subsequent process

of NF-κB2 (p100) precursor . Fourth, IKKα controls keratinocyte differentiation by a

kinase-independent mechanism that affects the production of keratinocyte

differentiation-inducing factor (kDIF) . IKKβ, in contrast, is not necessary for this

function. Finally, although both IKKα and IKKβ can phosphorylate multiple regions of

β-catenin, an opposite effect of IKKα and IKKβ on the transcriptional activity and

intracellular localization of β-catenin has been observed. Using mouse embryo

fibroblasts (MEF) lacking IKKα or IKKβ gene, Lamberti et al reported that IKKα

increased the nuclear localization and transcriptional activity of β-catenin, whereas

IKKβ decreased the nuclear localization and transcriptional activity of β-catenin.

Limited information is available concerning another recently identified IKK complex,

IKKi/ε. In contrast to the original IKK complex, this new IKK complex does not contain

IKKα, β or γ . IKKi/ε shares 27% homology with IKKα and IKKβ and possibly


mediates NF-κB-activating kinase (NAK) signaling, PMA/PKCε-induced S36

phosphorylation of IκBα, and NF-κB activation . In contrast to IKKα and IKKβ, which

are constitutively expressed in most cell types, the expression of IKKi/ε is inducible.

In the mouse macrophage cell line, RAW264.7, lipopolysaccharide (LPS) and some

other NF-κB-inducing cytokines, can drastically induce the accumulation of IKKi/ε

mRNA . Intriguingly, none of these inducers seem to be able to stimulate the kinase

activity of transfected IKKi/ε. Yeast two-hybrid screening by Nomura et al. recently

showed that the C- terminal portion of IKKi/ε can specifically associate with the N-

terminal domain of I-TRAF/TANK, an interaction protein of tumor necrosis factor

receptor-associated factor. Thus, it is possible that IKKi/ε either acts further upstream

of IKKα/β or at the same hierarchical level of IKKα/β after its association with I-

TRAF/TANK.

The predominant role of IKK is its activity as a serine/threonine kinase

phosphorylating IκB family proteins . Most of IκB family proteins contain a conserved

DSGXXS motif, where X is any amino acid . IKK is also able to phosphoyrlate NF-κB

p65 protein on a non-consensus site, S536 . The ability of IKK to exert its profound

kinase activity has led many intensive investigations to explore its likely role in other

cellular responses. Homology searches of the gene bank protein sequence database

reveal that a number of non-IκB/NF-κB family proteins also contain this motif. These

proteins include β-catenin, HIV vpu protein, phosphoinositide 3- kinase enhancer

(centaurin), c-Ski, Rho/Rac guanine nucleotide exchange factor, and a number of

other potential substrates listed in table 1. Except for β-catenin, which has been

recently demonstrated to be an IKK substrate , no experimental data so far suggests

that IKK can phosphorylate these non-IκB/NF-κB family proteins. New evidence

suggests that IKKα controls keratinocyte differentiation and IKKβ attenuates insulin

signaling related to type 2 diabetes and obesity . Thus, in addition to NF-κB signaling,

IKK might be involved in several other cellular signal transduction pathways in either

a kinase activity-dependent or a kinase activity-independent manner.

There are several good candidates for the inhibition of IKK activity. One group is the

nonsteroidal anti- inflammatory drugs (NSAIDs), including aspirin and sodium

salicylate. NSAIDs have been previously shown to inhibit activation of NF-κB and

cytokine-induced mRNA of cell adhesion molecules . Later studies suggested that

these effects of NSAIDs are the result of specific inhibition of ATP-binding to IKKβ ,


which is independent of their cyclooxygenase- 2 (COX-2) inhibitory activity. A second

candidate is the 15d- PGJ2, an anti-inflammatory cyclopentenone prostaglandin and

a natural ligand for peroxisome proliferator-activated receptor γ (PPARγ) . In Jurkat T

cells or HeLa cells, 15d-PGJ2 inhibits TPA- or TNFα-induced NF-κB activation in a

PPARγ-independent manner . A direct modification of cysteine 179 (C179) in the

activation loop of IKKβ by 15d-PGJ2 was observed in an in vitro IKK kinase activity

assay . The IC50 for inhibition of IKK activity was around 5 µM. The same

concentration of 15d- PGJ2, in contrast, stimulated JNK JNK activity, indicating that

15d-PGJ2 or its analogs may have therapeutic potential for diseases in which

inhibition of IKK and NF-κB may be desirable. However, it should be noted that the

inhibitory effect of 15d-PGJ2 on IKK and NF- κB might be cell type or stimulus

dependent. This becomes evident by the fact that 15d-PGJ2 potentiated LPS-induced

gene expression of IL-8, a NF-κB targeting gene . The third group of IKK inhibitors

includes several plant extracts that have been shown to reduce IKK activity in some

experimental systems. These extracts include resveratrol, parthenolide, and green

tea polyphenol (-)- epigallocatechin-3-gallate . The specificity and potential

application of these natural products in inhibiting IKK activity remain to be

investigated. Finally, two relatively specific IKK inhibitors are being developed by

Signal Pharmaceuticals and Novartis Pharma AG, respectively. Signal

Pharmaceuticals developed a selective IKKβ inhibitor named SPC839 that inhibits

IKKβ with nanomolar potency and IKKα with micromolar potency.

II. Upstream kinases of IKK

1. MEKK1

MEKK1 is a mammalian serine/threonine kinase in the mitogen-activated protein

kinase kinase kinase (MAPKKK) group. It was found that MEKK1 was a far more

important activator for JNK signaling rather than ERK signaling as was proposed

originally . The first evidence indicating the involvement of MEKK1 in signal-induced

IKK activation was provided by Lee et al. . In their studies, they reported that the

addition of the recombinant catalytic domain of MEKK1 (MEKK1∆ ) to the partially

enriched fraction of nonstimulated HeLa cells stimulated an IKK-like kinase activity

that phosphorylated IκBα at S32 and S36 and subsequent ubiquitination and

degradation of IκBα. Follow-up studies demonstrated that overexpression of MEKK1


stimulated the NF- κB-dependent transcriptional reporter . The activation of NF-κB by

HTLV Tax protein was shown to require MEKK1 . MEKK1 may also contribute to Toll-

and IL-1 receptor-mediated IKK activation, as demonstrated by an adaptor protein,

known as evolutionarily conserved signaling intermediate in Toll pathways (ECSIT)

that could promote the proteolytic activation of MEKK1 and subsequent activation of

NF-κB . Further studies by Mercurio et al. indicated the presence of a protein in the

IKK complex that is recognized by anti-MEKK1 antiserum .

Although MEKK1 has been shown to contribute to IKK activation in a number of

studies, the precise molecular link between these two kinases remains unclear. One

intriguing possibility is that MEKK1 may directly phosphorylate both IKKα and IKKβ at

the MAPKK activation loop, 176/177 S-X-X-X-S 180/181, where X is any amino acid.

Substitution of these serines with alanine residues inactivates both kinases, whereas

phosphomimetic glutamic acid substitution at these positions results in constitutively

active kinases . Nevertheless, it remains to be confirmed whether MEKK1 is a

physiological activator of IKK in cells in response to various stimuli. Indeed, a recent

study by Xia et al. demonstrated that inactivation of MEKK1 did not result in an

impairment of NF-κB activation in response to TNFα, IL-1, LPS, and dsRNA.

2. NF-κB-Inducing Kinase (NIK)

NIK, a member of the MAPKKK family, was originally identified as a tumor necrosis

factor (TNFα) receptor associating factor 2 (TRAF2)- interacting kinase whose

overexpression results in potent NF-κB activation without any considerable effect on

MAPKs . A study using yeast two-hybrid screens identified an interaction between

NIK and IKK, suggesting that NIK might be a direct upstream activator of IKK .

Transient transfection of NIK into human embryonic kidney 293 cells indicated that

IKKα was more responsive to NIK, whereas IKKβ was slightly more responsive to

MEKK1 . When the abilities of MEKK1 and NIK to activate total IKK kinase activity

are compared, most of studies show that NIK is a much stronger activator of the NF-

κB transcriptional reporter than MEKK1. NIK could preferentially phosphorylate IKKα

on Sl76 in the activation loop, leading to the activation of IKKα kinase activity. In

contrast, MEKK1 was found to preferentially phosphorylate the corresponding serine

residue, Sl77, in the activation loop of IKKβ. A dominant negative mutant of NIK

blocked NF- κB activation by TNFα, interleukin-1 (IL-1), FasFas, Toll-like receptors 2

and 4 , LMP1 and CD3/CD28 stimulation . Thus, NIK appears to be a general kinase


mediating IKK activation induced by diverse stimuli. However, a recent analysis using

the NIK-mutant mouse strain alymphoplasia (aly) contradicted this assumption. The

Alymohoplasia mouse strain failed to develop lymphoid organs, such as lymph nodes

and Peyer's patches due to a point mutation in the NIK locus . The mutation of NIK

locus results in disruption of interactions between NIK and IKKα or TRAF proteins.

Further analysis indicated that the aly mutation did not affect TNFα-induced

activation of NF- κB but only blocked lymphotoxin-mediated activation of NF-κB.

Similarly, studies using cells derived from NIK-deficient mice have indicated that NIK

appears to be dispensable in IKK activation induced by TNFα or IL-1 . Intriguingly,

lymphotoxin β induced a normal NF-κB DNA-binding activity in NIK-deficient cells,

whereas the same treatment failed to induce NF-κB reporter gene activity or NF-κB

target gene expression . It raises the possibility that NIK may be specifically involved

in IKK activation induced by lymphotoxin but not others.

3. NF-κB activating kinase (NAK)

Several groups independently identified a novel serine/threonine kinase, possibly

activating IKK through direct phosphorylation in cells stimulated with PMA . This

novel kinase was named NAK, TANK-binding-kinase 1 (TBK1), or T2K. Pomerantz

and Baltimore cloned NAK by a yeast two-hybrid screen using the N- terminal

stimulatory domain of TANK 1-190 fused to GAL4 as bait, and a human B-cell library

fused to the GAL4 activation domain. The same kinase was also identified by PCR

using degenerate primers based on sequences common to IKKα and IKKβ . The

amino acid sequence analysis indicated that the NAK protein contains a kinase

domain at its N-terminus that exhibits about 30% identity to the corresponding kinase

domains of IKKα and IKKβ, and more than 60% identity to the corresponding kinase

domain of IKKi/ε. The report by Pomerantz and Baltimore showed that NAK might

form a ternary complex with TRAF-2TANK and TRAF-2, suggesting that NAK

functions to the far more upstream of the signal cascade leading to IKK activation,

whereas in vitro kinase activation assay by Tojima and co-workers demonstrated that

NAK was a direct upstream kinase phosphorylating IKKβ. Interestingly, activation of

endogenous NAK resulted in only S36, but not S32 phosphorylation of IκBα, a similar

phenomenon observed in recombinant IKKi/ε- mediated IκBα phosphorylation . Since

both IKKα and IKKβ are able to phosphorylate both S32 and S36 of IκBα protein, it is

unclear whether IKKi/ε or a novel IKK isozyme functions as a downstream kinase of


NAK to induce S36 phosphorylation of IκBα. Transient transfection studies showed

that dominant negative NAK inhibited NF- κB transcriptional reporter activity induced

by PMA, PKCε, and PDGFPDGF, but not by TNFα, IL-1β, LPS, or ionizing radiation .

These results, therefore, suggest that NAK is likely to be a downstream kinase of

PKCε or related isozymes, and an upstream kinase of IKK in the signaling pathway

through which growth factors, such as PDGF, stimulate NF-κB activity.

4. Akt (PKB)

The pro-survival function of Akt has been well documented. The kinase activity of Akt

is activated via the phosphoinositide-3-OH kinase (Pl3K) and P13K-dependent

kinase 1/2 (PDKI/2) signaling pathways . Overexpression or constitutive activation of

Akt has been associated with tumorigenesis in a number of studies. As a

serine/threonine kinase, Akt is able to phosphorylate the pro-apoptotic protein

BadBad, the anti-apoptotic protein Bcl-xBcl-x, the apoptotic protease caspase-9, the

Forkhead transcription factors, and eNOS . However, considerable controversy

remains regarding the involvement of Akt in signal-induced IKK activation. Studies by

Ozes et al. and Xie et al. indicated that Akt was required for TNFα- or G protein

activator-induced NF-κB activation by directly phosphorylating and activating IKKα in

293, HeLa, and ME-180 cells. A putative Akt phosphorylation site at amino acids 18

to 23 in both IKKα and IKKβ was identified. Akt induced T23 phosphorylation of IKKα

both in vitro and in vivo. Mutation of T23 significantly decreased Akt-induced IKKα

phosphorylation and TNFα-induced NF-κB activation in 293, HeLa, and ME-180 cells

. By contrast, Romashkova et al. demonstrated that Akt was involved in PDGF-

mediated, but not TNFα− or PMA-mediated NF-κB activation in human or rat

fibroblasts. In this study, the authors suggested that upon PDGF stimulation, Akt

could transiently associate with IKK and induce the activation IKK, especially IKKβ.

Several other studies, however, contradicting these reports, suggested that the

effects of Akt on NF-κB did not occur at the level of IKK activation in several cell

types. A study by Delhase and co- workers indicated that Akt activation induced by

IGF-I failed to activate IKKα, IκBα phosphorylation and degradation, or NF-κB DNA

binding in HeLa cells, the same cell line used by Ozes et al . Similarly, several recent

studies showed that Akt was not involved in TNFα-induced NF-κB activation in

human vascular smooth muscle cells, skin fibroblasts, or endothelial cells . Rather,

Akt might enhance the ability of the p65 (RelA) transactivation to induce transcription


. In Jurkat T-cells, Akt alone failed to activate NF-κB, but it was capable of

potentiating NF-κB activation induced by PMA, partially by enhancing

IκBβ degradation . Evidence further supporting this notion is the observation that

expression of constitutively active Akt upregulates the mRNA level of β-TrCP, a

subunit of the SCF-β-TrCP complex responsible for the ubiquitination of IκBα or IκBβ

proteins . Thus, it is possible that Akt phosphorylates IKK in a cell context- and

stimulation-dependent manner. It is also highly possible that several different

mechanisms are involved in Akt-regulated NF-κB activation. One remaining question

is whether or not upstream kinases of Akt, such as PDK1 and PDK2, also activate

IKK, since both IKKα and IKKβ contain a putative PDK1 phosphorylation site (S-F-X-

G-T-X-X-Y-X-A-P-E) directly juxtaposed to the MAPKKK phosphorylation site .

5. Mixed-Lineage Kinase 3 (MLK3)

MLK3, another member of the MAPKKK family, contains an N-terminal SH3 domain,

followed by the catalytic domain and two tandem leucine/isoleucine zippers, a basic

region, a Cdc42/Rac binding motif, and a proline-rich C terminus . Based on these

structural characteristics, MLK3 may associate with a variety of protein modules.

Studies by Hehner et al. suggested that MLK3 could directly associate with IKK

complex through its leucine zipper domain and phosphorylate Sl76 of IKKα and Sl77

and Sl8l of IKKβ. Transfection of Jurkat T cells with a kinase- mutated form of MLK3

blocked CD3-CD28 signal- and PMA-induced NF-κB transcriptional activity. No

significant influence of this mutated MLK3 was observed on either TNFα- or IL-1-

induced NF-κB activation. These results suggest that MLK3 may be important in

mediating T-cell co-stimulation-induced activation of IKK and consequent NF-κB-

dependent transcription. MLK3 has also been shown to form a complex with a JNK

scaffold protein JIP and stimulate JNK activation . Thus, MLK3 may function as an

integral molecule between the signaling pathways leading to the activation of NF-κB

and JNK, which would provide a molecular explanation why many stimuli induce NF-

κB and JNK simultaneously under certain circumstances.

6. TGFβ-Activated Kinase 1 (TAK1)

TAK1 is a member of the MAPKKK (MAP3K) family, which was originally identified as

a kinase mediating the signaling pathway of TGFβ superfamily members .

Transfection of the cells with an activated form of TAK1, in which the N-terminal 22

amino acids are deleted, induces expression of a reporter gene governed by TGFβ-


responsive promoter . However, the biochemical link between TGFβ and TAK1 has

been elusive. Ironically, the contributions of TAK1 to signal-induced NF-κB and JNK

activation have been studied intensively. Several new insights into the roles of TAK1

in IKK activation have emerged in the cellular response to cytokines or Toll signals .

The first evidence to suggest that TAK1 is involved in NF- κB signaling came from

studies in which overexpression of TAK1 together with its activator protein, TAK1

binding protein 1 (TAB1), induced the nuclear translocation of NF- κB in a NIK-

independent manner . Further studies indicated a direct physiological interaction

between TAK1 and IKK in unstimulated cells . Recruitment of TAB1 and/or TAB2 to

TAK1 activates the kinase activity of TAK1, resulting in phosphorylation of the serine

residues in the activation loop of IKK and subsequent dissociation of TAK1 from IKK

complex . In Drosophila, a null mutation in the TAK1 gene produces phenotypes

similar to that of mutations in immune deficiency (Imd), and IKK, suggesting that

TAK1 is a direct kinase mediating Imd signals . Genetic studies and sequential

chromatographic purification by Wang et al. demonstrate that the kinase activity of

TAK1 in response to IL-1 is dependent on the TRAF6 protein, which has been

modified by a distinct polyubiquitin chain assembled through the lysine 63 (K63) at

each ubiquitin molecules. In contrast to the polyubiquitin chain in which the C-

terminal glycine 76 (G76) of one ubiquitin is ligated to the K48 side chain of the

neighboring ubiquitin, the polyubiquitin chain linked through G76-K63 does not target

proteins for proteasomal degradation, but rather, activates the function of proteins

(see below) . The major debatable issues in TAK1-induced IKK activation are the

involvement and hierarchical position of NIK. Whereas several reports clearly

suggest that NIK is not involved in TAK1-induced IKK activation and TAK1 is a direct

upstream kinase phosphorylating IKK in HeLa cells treated with IL-1 , Ninomiya-Tsuji

et al. showed that NIK is a mediator of TAK1-induced IKK activation in IL-1-treated

293 cells. It is unclear whether this discrepancy is due to the cell type or subtle

differences in overexpression of the dominant-negative inactive NIK mutant.

7. Other Kinases

A variety of other kinases have been reported to function upstream of IKK. Because

of the lack of evidence of direct association of these kinases with IKK upon activation

or specific phosphorylation site(s) of these kinases on IKK, it is unclear whether these

kinases are direct upstream kinases phosphorylating and activating of IKK, or far


more distal kinases indirectly activating IKK. These kinases include Cot ,

PKCζ, PKCα , PKCθ , or PKR etc. In light of the fact that a variety of kinases can

affect IKK, it seems likely that different cell types and stimuli may utilize distinct

upstream kinases for the activation of IKK. An example to support this notion is the

observation that PKCθ and Cot kinase participate in CD3-CD28 costimulation signal-

induced, but not TNFα- induced, activation of NF-κB .

III. Mechanisms of Ubiquitination in NF-κB Activation

As detailed above, the activation of NF-κB by most of the extracellular inducers is

dependent on the phosphorylation and subsequent degradation of IκB proteins. A

crucial step during this process is the phosphorylation-dependent conjugation of IκB

proteins with polyubiquitin chain, a marker required for the proteasomal degradation

of IκBα. Whereas the ubiquitination sites on IκBβ and IκBε have not been definitely

identified , lysines 21 and 22 (K21 and K22) on the IκBα protein were considered as

the major sites conjugated by the polyubiquitin chain .

Figure 2 : SCF-β-TrCP ubiquitin ligase complex-mediated ubiquitination of IκB proteins. The basic

components of this E3 complex include Skp1, Cul-1 (CDC53), and the F-box protein, β-TrCP. β-TrCP

recognizes and links the phosphorylated IκB proteins to this complex allowing the ubiquitination of

IκBs by ubiquitin-conjugating enzyme E2 following the C-terminal G residue activation of ubiquitin by

ubiquitin- activating enzyme E1. Ubiquitin is a highly conserved and heat stable 76- amino acid protein found in

virtually all types of eukaryotic cells . Ubiquitination of proteins involves three or four

sequential steps (Fig. 2). Initially, the C-terminal glycine (G76) of ubiquitin is activated

by ATP to form a high energy thiolester intermediate catalyzed by the ubiquitin-

activating enzyme (Uba or E1). Activated ubiquitin is then transferred from E1 to one

of many distinct ubiquitin-conjugating enzymes (Ubc or E2), forming a similar

thioester-linked complex. Finally, with the aid of ubiquitin ligases (E3), an isopeptide

bond is formed between the activated C-terminal G76 of ubiquitin and an ε-NH2


group of a K residue of the substrate. In successive reactions, polyubiquitin chain, is

synthesized by progressive transfer of ubiquitin moieties to K48 or K63 of the

previously conjugated ubiquitin molecule, forming G76-K48 or G76-K63 isopeptide

bonds. An assembly factor, named Ufd or E4, may be required for this process . The

specificity of protein ubiquitination is usually determined by the ubiquitin ligase E3

that recognizes specific substrates. At least three types of ubiquitin ligase E3

complexes have been well documented. These ligase complexes include the Skp1-

cullin-F-box (SCF) complex, the VHL protein-elongin B-elongin complex (VBC), and

the anaphase promoting complex (APC). The ubiquitin ligase E3, responsible for the

ubiquitination of IκBα, is the SCF complex containing a F-box/Trp-Asp repeating

(WD) protein named β-TrCP (Fig. 2). Following phosphorylation of S32 and S36 in

the conserved DSGXXS motif of IκBα by IKK, the β-TrCP subunit of the SCF

complex recognizes and binds to the phosphorylated DSGXXS motif of IκBα . The

binding of SCF to I κBα results in the association of SCF with specific E2s, including

Ubc3, Ubc4, Ubc5, and Ubc9 . These E2s are able to catalyze the ubiquitin

conjugation of IκBα and the assembly of G76-K48 polyubuquitin chain. Consistent

with the likely role for SCF-β-TrCP as a ubiquitin ligase complex conjugating

polyubiquitin chain to IκBα, is the observation that Slimb protein, a Drosophila

homology of mammalian β-TrCP, is required for the ubiquitination of Cactus, an IκB-

like protein inhibiting the activation of the Drosophila NF-κB homolog, Dorsal . During

dorsoventral patterning of the early Drosophila embryo, the Dorsal protein is

activated specifically on the ventral side of the embryo by the Toll receptor-signaling

pathway. These findings point to the existence of an evolutionarily conserved

pathway for specific ubiquitination of the IκBα protein for the purpose of dynamic

signal transduction from the receptor to NF-κB.

In parallel studies of signal-induced IκBα ubiquitination, several reports indicated that

this process could be antagonized by SUMO-1 (small ubiquitin-related modifier-1)

modification of IκBα on the same residues where the polyubiquitin chain is

conjugated , or by unknown product(s) of nonpathogenic Salmonella bacteria .

SUMO-1 is one of the best-characterized members of ubiquitin-related proteins.

Conjugation of SUMO-1 to substrates requires SUMO-1-activating enzyme

Aos/Uba2, and SUMO-1-conjugating enzyme, Ubc9. Although substrates can be

modified by several SUMO-1 at distinct sites, no multi-SUMO-1 chains are apparently


formed . In contrast to ubiquitination of IκBα protein, SUMO-1 conjugation does not

target IκBα to proteasomal degradation . The inhibition of NF-κB by certain bacterial

pathogens may be through a mechanism affecting the conjugation of SUMO-1 on IκB

proteins. One example is the observation that YopJ, a protein product encoded by a

70-kB plasmid harbored in the Yersinia species that caused the Black Death in the

Middle Ages, inhibits MEKK1-induced NF-κB activation. Earlier study by Orth et al.

indicated that the inhibition of NF-κB by YopJ is through direct interaction of YopJ

with IKKβ but not with IKKα. Structural analysis of YopJ protein by the same group

later suggested that YopJ might be a SUMO-protease promoting the conversion of

precursor SUMO-1 to mature SUMO-1 . Nevertheless, whether YopJ enhances the

conjugation of SUMO-1 on IκBα has not been demonstrated.

The vast majority of ubiquitination reactions in which the proteins are ubiquitinated

via G76-K48 assembly of the polyubiquitin chain target protein for proteasomal

degradation. Examples include the ubiquitination of IκBα, p53, cyclins, c- Jun, and

others. This is not, however, the case of ubiquitination of proteins via G76-K63

assembly of polyubiquitin chain. The biochemical evidence of G76-K63 assembly of

the polyubiquitin chain remains elusive, but it appears to be independent of

proteasomal degradation . Recent studies by Wang et al. suggest that linkage of the

G76-K63 polyubiquitin chain with TRAF6 protein plays an important role in mediating

TLR/IL-1R signal-induced activation of TAK1, an upstream kinase of IKK. TRAF6

itself exhibits the ubiquitin ligase E3 activity by the structural characteristic of RING

fingers in its C-terminus.

The nature of the upstream regulators that promote G76-K63 ubiquitination of TRAF6

is less clear. One good candidate, however, is the Ubc complex composed of Ubc13,

a member of the ubiquitin-conjugating enzyme E2 family, and Uev1A, a ubiquitin-

conjugating E2 enzyme variant . In yeast and mammalian cells, both Ubc13 and

Uev1A are considered the major enzymes required for the synthesis of G76-K63

polyubiquitin . In chromatographic purification of HeLa cell cytoplasmic extracts, the

Ubc13/Uev1A complex was found to be co-eluted with TRAF6 and appeared to be

essential in TRAF6- induced TAK1 and subsequent IKK activation . However, in

Drosophila, this Ubc13/Uev1A-induced K63 polyubiquitination of TRAF6 has yet to be

established, despite the identification of the Drosophila homologs of Ubc13 and

Uev1A, bendless and dUev1A, respectively . Interestingly, Ubc13/Uev1A was also


found to interact genetically with a DNA repair protein, Rad5 , indicating that it is

likely to be coupled to a number of cellular processes. Such a finding supports the

likelihood that the activation of the Ubc complex provides a mechanism by which IKK

signals can be selectively activated during cellular damage response in vivo.

IV. ROIs: Critical Mediators or bystanders in NF-κB Activation?

Oxidative stress is a hallmark of pathophysiological response resulting from the

alterations of cellular redox homoeostasis due to either an over-production of reactive

oxygen intermediates (ROIs) or a deficiency in buffering or scavenging system for

ROIs . Typically, the oxidatively stressed cells exhibit damage of their

macromolecules leading to lipid peroxidation, oxidation of amino acid side chains

(especially cysteine), DNA damage, stress response kinase activation and gene

expression associated with cell cycle arrest and/or cell apoptosis. Moderate oxidative

stress without severe damage of structural and functional macromolecules can be

recovered due to the activation of cellular defense systems including nonenzymatic

and enzymatic antioxidants. A number of stress response genes are induced to

protect cells from the oxidative stress or to repair the ROI-mediated damages. A

sustained oxidative stress produced during chronic or acute inflammatory response

and/on environmental toxicant exposure, however, will be cytotoxic.

Among all the known oxidative stress inducers, H2O2 and some environmental toxic

metals or particles are perhaps the most potent and well studied . Many other agents,

such as TNFα, IL-1 and bacterial or viral proteins, also induce oxidative stress .

Since the discovery of NF-κB, hundreds of reports have indicated that some

extracellular stimuli that induce oxidative stress also activate NF-κB . Thus, it is not

too surprising that many researchers attributed a role for ROIs in signal- induced NF-

κB activation . Some even proposed that ROIs might be universal molecules

mediating the activation of NF-κB in response to a broad range of stimuli . However,

the conclusion that ROIs mediate NF-κB activation has been strongly challenged .

First, correlations between ROI generation and NF-κB activation do not necessarily

mean ROIs are essential mediators linking upstream signals to NF-κB activation.

Under certain circumstances, ROI generation may be simply a bystander signal or a

secondary response to NF-κB activation. Second, caution should be exercised in

interpreting the inhibitory effects of a various antioxidants on signal-induced NF-κB.

Many antioxidants can disturb the normal cellular redox status that maintains the


basal signaling potential required for the activation of NF-κB or other intracellular

biochemical events even under the non-oxidative stress conditions. In addition, many

low-molecular-weight antioxidants may inhibit NF-κB by non-antioxidant actions .

Third, it should be noted that several studies show that ROIs fail to activate NF-κB in

many experimental systems . Finally, emerging evidence suggests that the DNA

binding activity of activated and nuclear translocated NF-κB requires reducing

conditions . Oxidation or nitrosylation of the cysteine residue in the DNA binding

domain of the NF-κB p50 subunit suppresses the DNA binding and transcriptional

activity of NF-κB .

The signal transduction pathway, such as the upstream and proximal kinases,

leading to the activation of NF-κB by TNF, IL-1, Toll, LPS, and CD28, has been

clearly identified. However, only limited information is available to suggest the

responsiveness of these kinases to ROIs . The evidence to implicate ROIs as

stimulators of IKK is based on the elevated IKK activity in human epithelial cells or

mouse fibroblast cells by the H2O2 treatment . In our own studies, we found a modest

induction of IKK activity in cellular response to chromium(VI), a potent intracellular

H2O2 inducer (Chen et al., unpublished). Nevertheless, studies challenging this

observation exist. Li and Karin could not detect IKK kinase activity in HeLa cells

stimulated with UV-C, another intracellular H2O2 inducer , despite the fact that UV-C

induced IκBα degradation and NF-κB DNA binding. Similarly, Korn et al. described

that H2O2 itself failed to stimulate IKK, but rather, inhibited TNFα-induced IKK activity.

It is highly likely that H2O2 inactivates IKK through direct oxidation of a conserved

cysteine 179 (C179) in the kinase domain of IKKβ, a mechanism similar to the

inactivation of IKKβ by 15d-PGJ2 and a high concentration of arsenic .


Figure 3 : Model for the ROI-induced oxidation of Trx and the subsequent ubiquitination of IκBs.

Oxidation of the C-X-X-C motif of Trx induces its dissociation from ASK1, thereby allowing the

dimerization and activation of ASK1. JNK is activated by SEK1 that has been phosphorylated by

ASK1, leading to the accumulation of β-TrCP which is required for the processes of IκB ubiquitination. Whereas IKK seems to be a less favorable target- point in ROI-modulated NF-κB

activation, kinases other than IKK may serve as bridge molecules linking ROIs to the

activating signals of NF-κB. One such kinase, JNK, merits special attention, not only

because of its unequivocal activation in response to ROIs, but also because of its

potential link to the ubiquitination and subsequent degradation of IκBα . The

activation of JNK by ROIs appears to be mediated by the activation of ASK1, a

member of the MAPKKK family that phosphorylates and activates SEK1 (MKK4), an

upstream kinase of JNK (Fig. 3). In resting cells, ASK1 binds with high affinity to the

reduced form of thioredoxin (Trx) which serves as an inhibitor of ASK1 by preventing

the dimerization of ASK1 . Oxidation of the C-X-X-C motif of Trx by ROIs induces the

dissociation of Trx from ASK1, thereby allowing the dimerization of ASK1 and the

consequent activation of JNK. We have previously reported that inhibition of JNK by

overexpression of a dominant negative SEK1 impaired the degradation of IκBα and

the activation of NF-κB induced by vanadate . This observation was further

substantiated by Spiegelman et al. who provided convincing evidence indicating the

contribution of JNK to the signal-induced ubiquitination of IκBα protein. Activation of

JNK resulted in accumulation of β-TrCP, a subunit of the SCF-β-TrCP complex that


recognizes the phosphorylates DSGXXS motif within the IκBα protein and causes the

subsequent ubiquitination . While JNK has been implicated in the stabilization of a

number of short-lived mRNAs in response to stress , it is plausible to speculate that

the JNK-mediated accumulation of β-TrCP is through stabilization of the β-TrCP

mRNA. Indeed, analysis of the β-TrCP mRNA sequence by Spiegelman et al.

revealed a closely resembled JNK response element in addition to two AU-rich

elements in the 3’-UTR region of β-TrCP mRNA. Since ubiquitination of the IκBα

protein is potentially a rate-limiting step, the abundance of β-TrCP regulated by JNK

may serve as an important point of regulation in ROI-induced NF-κB activation.

Table 1: Confirmed and putative IKK substrates in mammalian cells.

Substrates Consensus sites References

Confirmed substrates:

IkBa 31 D S G L D S 3

IkBb 18 D S G L G S 3

IkBe 17 D S G I E S 3

70 D S T Y G S

p105 921 D S V C D S 22

925 D S G V E T 23

p65 535 S S I A D M 24

b-Catenin 32 D S G I H S 10

Putative substrates:

Rho-GEF 220 D S G L D S

CDC2-related kinase 7 439 D S G L E S

Centaurin b2 386 D S G N E S

HNF-3a 35 N S G L G S

Metastasis suppressor protein 321 D S G F I S

PPARg coactivator 1 60 D S G F V S

TRAF6 285 D S G Y I S

hIRS-1 423 D g G F I S

Plakoglobin 23 D S G I H S

Carboxypeptidase A1 175 D T G I H S


C-Met oncogene 284 DS G L H S

RAD51 associated protein 1 18 D S G N D S

Chromodomain helicase 53 D S G S E S

TNFR2 72 D T V C D S

Tight junction protein 1 (ZO1) 810 D S G V E T

Pannexin 2 265 D S G V Q T

Eps8 809 D S G V E S

Summary

Increasing evidence implicates dysregulation of signaling pathways leading to the

activation of the NF-κB transcription factor in the pathology of various diseases,

including autoimmune diseases, neurodegenerative diseases, inflammation, and

cancers . Moreover, several human diseases due to the inherited mutations in the

genes encoding NF-κB signaling molecules have been recently described . The

signal transduction pathways of NF-κB activation therefore represent potential targets

for therapeutic intervention. As discussed above, tremendous advances have been

made in our understanding of the upstream signaling pathways of NF-κB activation.

Yet this understanding is not complete. A fundamental goal for future studies is to

focus on the structural and functional aspects of the participating components in

these pathways. It seems likely that new aspects of NF-κB signaling will be

discovered through such studies. 1Abbreviations:

IKK, IκB kinase complex; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated

protein kinases; ROIs, reactive oxygen intermediates; NIK, NF-κB inducing kinase;

NAK, NF-κB activating kinase; TAK1, TGF-β activating kinase 1; MLK, mixed lineage

kinase.

Acknowledgments

We thank our colleague Drs Murali Rao and Vince Castranova for helpful

suggestions and critique of the manuscript. We apologize to those whose primary

valuable work was not cited because of space limitation. Dr. Fei Chen thanks the

Health Effects Laboratory Division/National Institute for Occupational Safety and

Health for its continuing supporting.

1. Sen, R., and Baltimore, D. (1986) Cell 47(6), 921-8. Medline


2. Chen, F., Castranova, V., and Shi, X. (2001) Am J Pathol 159(2), 387-97. 3. Karin, M., and Ben-Neriah, Y. (2000) Annu Rev Immunol 18, 621-63 4. Kopp, E. B., and Ghosh, S. (1995) Adv Immunol 58, 1-27 5. Baldwin, A. S., Jr. (1996) Annu Rev Immunol 14, 649-83 6. Beg, A. A., and Baldwin, A. S., Jr. (1993) Genes Dev 7(11), 2064-70. 7. Israel, A. (2000) Trends Cell Biol 10(4), 129-33. 8. Peters, R. T., and Maniatis, T. (2001) Biochim Biophys Acta 2(62), M57-62 9. Silverman, N., and Maniatis, T. (2001) Genes Dev 15(18), 2321-42. 10. Lamberti, C., Lin, K. M., Yamamoto, Y., Verma, U., Verma, I. M., Byers, S., and

Gaynor, R. B. (2001) J Biol Chem 29, 29 11. Kroll, M., Margottin, F., Kohl, A., Renard, P., Durand, H., Concordet, J. P.,

Bachelerie, F., Arenzana-Seisdedos, F., and Benarous, R. (1999) J Biol Chem 274(12), 7941-5.

12. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J., Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997) Science 278(5339), 860-6.

13. Woronicz, J. D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D. V. (1997) Science 278(5339), 866-9.

14. Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91(2), 243-52.

15. Nakano, H., Shindo, M., Sakon, S., Nishinaka, S., Mihara, M., Yagita, H., and Okumura, K. (1998) Proc Natl Acad Sci U S A 95(7), 3537-42.

16. Senftleben, U., Cao, Y., Xiao, G., Greten, F. R., Krahn, G., Bonizzi, G., Chen, Y., Hu, Y., Fong, A., Sun, S. C., and Karin, M. (2001) Science 293(5534), 1495-9.

17. Hu, Y., Baud, V., Oga, T., Kim, K. I., Yoshida, K., and Karin, M. (2001) Nature 410(6829), 710-4.

18. Peters, R. T., Liao, S. M., and Maniatis, T. (2000) Mol Cell 5(3), 513-22. 19. Shimada, T., Kawai, T., Takeda, K., Matsumoto, M., Inoue, J., Tatsumi, Y.,

Kanamaru, A., and Akira, S. (1999) Int Immunol 11(8), 1357-62. 20. Pomerantz, J. L., and Baltimore, D. (1999) EMBO J 18(23), 6694-704. 21. Nomura, F., Kawai, T., Nakanishi, K., and Akira, S. (2000) Genes Cells 5(3), 191-

202. 22. Heissmeyer, V., Krappmann, D., Wulczyn, F. G., and Scheidereit, C. (1999) EMBO J

18(17), 4766-78. 23. Salmeron, A., Janzen, J., Soneji, Y., Bump, N., Kamens, J., Allen, H., and Ley, S. C.

(2001) J Biol Chem 276(25), 22215-22. 24. Sakurai, H., Chiba, H., Miyoshi, H., Sugita, T., and Toriumi, W. (1999) J Biol Chem

274(43), 30353-6. 25. Yuan, M., Konstantopoulos, N., Lee, J., Hansen, L., Li, Z. W., Karin, M., and

Shoelson, S. E. (2001) Science 293(5535), 1673-7. vKopp, E., and Ghosh, S. (1994) Science 265(5174), 956-9.

26. Yin, M. J., Yamamoto, Y., and Gaynor, R. B. (1998) Nature 396(6706), 77-80. 27. Yamamoto, Y., Yin, M. J., Lin, K. M., and Gaynor, R. B. (1999) J Biol Chem

274(38), 27307-14. 28. Ricote, M., Li, A. C., Willson, T. M., Kelly, C. J., and Glass, C. K. (1998) Nature

391(6662), 79-82. 29. Jiang, C., Ting, A. T., and Seed, B. (1998) Nature 391(6662), 82-6. 30. Rossi, A., Elia, G., and Santoro, M. G. (1997) Proc Natl Acad Sci U S A 94(2), 746-

50.


31. Rossi, A., Kapahi, P., Natoli, G., Takahashi, T., Chen, Y., Karin, M., and Santoro, M. G. (2000) Nature 403(6765), 103-8.

32. Zhang, X., Wang, J. M., Gong, W. H., Mukaida, N., and Young, H. A. (2001) J Immunol 166(12), 7104-11.

33. Holmes-McNary, M., and Baldwin, A. S., Jr. (2000) Cancer Res 60(13), 3477-83. 34. Kwok, B. H., Koh, B., Ndubuisi, M. I., Elofsson, M., and Crews, C. M. (2001) Chem

Biol 8(8), 759-66. 35. Yang, F., Oz, H. S., Barve, S., de Villiers, W. J., McClain, C. J., and Varilek, G. W.

(2001) Mol Pharmacol 60(3), 528-33. 36. Minden, A., Lin, A., McMahon, M., Lange-Carter, C., Derijard, B., Davis, R. J.,

Johnson, G. L., and Karin, M. (1994) Science 266(5191), 1719-23. 37. Chang, L., and Karin, M. (2001) Nature 410(6824), 37-40. 38. Lee, F. S., Hagler, J., Chen, Z. J., and Maniatis, T. (1997) Cell 88(2), 213-22. 39. Li, X. H., Murphy, K. M., Palka, K. T., Surabhi, R. M., and Gaynor, R. B. (1999) J

Biol Chem 274(48), 34417-24. 40. Kopp, E., Medzhitov, R., Carothers, J., Xiao, C., Douglas, I., Janeway, C. A., and

Ghosh, S. (1999) Genes Dev 13(16), 2059-71. 41. Xia, Y., Makris, C., Su, B., Li, E., Yang, J., Nemerow, G. R., and Karin, M. (2000)

Proc Natl Acad Sci U S A 97(10), 5243-8. 42. Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature

385(6616), 540-4. 43. Ling, L., Cao, Z., and Goeddel, D. V. (1998) Proc Natl Acad Sci U S A 95(7), 3792-7. 44. Cohen, L., Henzel, W. J., and Baeuerle, P. A. (1998) Nature 395(6699), 292-6. 45. Kirschning, C. J., Wesche, H., Merrill Ayres, T., and Rothe, M. (1998) J Exp Med

188(11), 2091-7. 46. Muzio, M., Natoli, G., Saccani, S., Levrero, M., and Mantovani, A. (1998) J Exp Med

187(12), 2097-101. 47. Sylla, B. S., Hung, S. C., Davidson, D. M., Hatzivassiliou, E., Malinin, N. L.,

Wallach, D., Gilmore, T. D., Kieff, E., and Mosialos, G. (1998) Proc Natl Acad Sci U S A 95(17), 10106-11.

48. Lin, X., Cunningham, E. T., Jr., Mu, Y., Geleziunas, R., and Greene, W. C. (1999) Immunity 10(2), 271-80.

49. Shinkura, R., Kitada, K., Matsuda, F., Tashiro, K., Ikuta, K., Suzuki, M., Kogishi, K., Serikawa, T., and Honjo, T. (1999) Nat Genet 22(1), 74-7.

50. Yin, L., Wu, L., Wesche, H., Arthur, C. D., White, J. M., Goeddel, D. V., and Schreiber, R. D. (2001) Science 291(5511), 2162-5.

51. Tojima, Y., Fujimoto, A., Delhase, M., Chen, Y., Hatakeyama, S., Nakayama, K., Kaneko, Y., Nimura, Y., Motoyama, N., Ikeda, K., Karin, M., and Nakanishi, M. (2000) Nature 404(6779), 778-82.

52. Khwaja, A. (1999) Nature 401(6748), 33-4. 53. Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., and Donner, D.

B. (1999) Nature 401(6748), 82-5. 54. Xie, P., Browning, D. D., Hay, N., Mackman, N., and Ye, R. D. (2000) J Biol Chem

275(32), 24907-14. 55. Romashkova, J. A., and Makarov, S. S. (1999) Nature 401(6748), 86-90. 56. Delhase, M., Li, N., and Karin, M. (2000) Nature 406(6794), 367-8. 57. Rauch, B. H., Weber, A., Braun, M., Zimmermann, N., and Schror, K. (2000) FEBS

Lett 481(1), 3-7. 58. Madge, L. A., and Pober, J. S. (2000) J Biol Chem 275(20), 15458-65.


59. Madrid, L. V., Wang, C. Y., Guttridge, D. C., Schottelius, A. J., Baldwin, A. S., Jr., and Mayo, M. W. (2000) Mol Cell Biol 20(5), 1626-38.

60. Sizemore, N., Leung, S., and Stark, G. R. (1999) Mol Cell Biol 19(7), 4798-805. 61. Kane, L. P., Shapiro, V. S., Stokoe, D., and Weiss, A. (1999) Curr Biol 9(11), 601-4. 62. Spiegelman, V. S., Slaga, T. J., Pagano, M., Minamoto, T., Ronai, Z., and Fuchs, S. Y.

(2000) Mol Cell 5(5), 877-82. 63. Peterson, R. T., and Schreiber, S. L. (1999) Curr Biol 9(14), R521-4. 64. Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S. C., Hemmings, B.

A., and Thomas, G. (1998) Science 279(5351), 707-10. 65. Teramoto, H., Coso, O. A., Miyata, H., Igishi, T., Miki, T., and Gutkind, J. S. (1996) J

Biol Chem 271(44), 27225-8. 66. Hehner, S. P., Hofmann, T. G., Ushmorov, A., Dienz, O., Wing-Lan Leung, I.,

Lassam, N., Scheidereit, C., Droge, W., and Schmitz, M. L. (2000) Mol Cell Biol 20(7), 2556-68.

67. Yamaguchi, K., Shirakabe, K., Shibuya, H., Irie, K., Oishi, I., Ueno, N., Taniguchi, T., Nishida, E., and Matsumoto, K. (1995) Science 270(5244), 2008-11.

68. Vidal, S., Khush, R. S., Leulier, F., Tzou, P., Nakamura, M., and Lemaitre, B. (2001) Genes Dev 15(15), 1900-12.

69. Takaesu, G., Kishida, S., Hiyama, A., Yamaguchi, K., Shibuya, H., Irie, K., Ninomiya-Tsuji, J., and Matsumoto, K. (2000) Mol Cell 5(4), 649-58.

70. Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., Slaughter, C., Pickart, C., and Chen, Z. J. (2000) Cell 103(2), 351-61.

71. Ninomiya-Tsuji, J., Kishimoto, K., Hiyama, A., Inoue, J., Cao, Z., and Matsumoto, K. (1999) Nature 398(6724), 252-6.

72. Sakurai, H., Miyoshi, H., Toriumi, W., and Sugita, T. (1999) J Biol Chem 274(15), 10641-8.

73. Wang, C., Deng, L., Hong, M., Akkaraju, G. R., Inoue, J., and Chen, Z. J. (2001) Nature 412(6844), 346-51.

74. Sakurai, H., Shigemori, N., Hasegawa, K., and Sugita, T. (1998) Biochem Biophys Res Commun 243(2), 545-9.

75. Hofmann, R. M., and Pickart, C. M. (2001) J Biol Chem 276(30), 27936-43. 76. Lallena, M. J., Diaz-Meco, M. T., Bren, G., Paya, C. V., and Moscat, J. (1999) Mol

Cell Biol 19(3), 2180-8. 77. Sun, Z., Arendt, C. W., Ellmeier, W., Schaeffer, E. M., Sunshine, M. J., Gandhi, L.,

Annes, J., Petrzilka, D., Kupfer, A., Schwartzberg, P. L., and Littman, D. R. (2000) Nature 404(6776), 402-7.

78. Lin, X., O'Mahony, A., Mu, Y., Geleziunas, R., and Greene, W. C. (2000) Mol Cell Biol 20(8), 2933-40.

79. Zamanian-Daryoush, M., Mogensen, T. H., DiDonato, J. A., and Williams, B. R. (2000) Mol Cell Biol 20(4), 1278-90.

80. Wu, C., and Ghosh, S. (1999) J Biol Chem 274(42), 29591-4. 81. Shirane, M., Hatakeyama, S., Hattori, K., and Nakayama, K. (1999) J Biol Chem

274(40), 28169-74. 82. Weil, R., Laurent-Winter, C., and Israel, A. (1997) J Biol Chem 272(15), 9942-9. 83. Baldi, L., Brown, K., Franzoso, G., and Siebenlist, U. (1996) J Biol Chem 271(1),

376-9. 84. Chen, Z., Hagler, J., Palombella, V. J., Melandri, F., Scherer, D., Ballard, D., and

Maniatis, T. (1995) Genes Dev 9(13), 1586-97. 85. Pickart, C. M. (2001) Annu Rev Biochem 70, 503-533.


86. Koegl, M., Hoppe, T., Schlenker, S., Ulrich, H. D., Mayer, T. U., and Jentsch, S. (1999) Cell 96(5), 635-44.

87. Winston, J. T., Strack, P., Beer-Romero, P., Chu, C. Y., Elledge, S. J., and Harper, J. W. (1999) Genes Dev 13(3), 270-83.

88. Hatakeyama, S., Kitagawa, M., Nakayama, K., Shirane, M., Matsumoto, M., Hattori, K., Higashi, H., Nakano, H., Okumura, K., Onoe, K., Good, R. A., and Nakayama, K. (1999) Proc Natl Acad Sci U S A 96(7), 3859-63.

89. Strack, P., Caligiuri, M., Pelletier, M., Boisclair, M., Theodoras, A., Beer-Romero, P., Glass, S., Parsons, T., Copeland, R. A., Auger, K. R., Benfield, P., Brizuela, L., and Rolfe, M. (2000) Oncogene 19(31), 3529-36.

90. Tashiro, K., Pando, M. P., Kanegae, Y., Wamsley, P. M., Inoue, S., and Verma, I. M. (1997) Proc Natl Acad Sci U S A 94(15), 7862-7.

91. Spencer, E., Jiang, J., and Chen, Z. J. (1999) Genes Dev 13(3), 284-94. 92. Desterro, J. M., Rodriguez, M. S., and Hay, R. T. (1998) Mol Cell 2(2), 233-9. 93. Neish, A. S., Gewirtz, A. T., Zeng, H., Young, A. N., Hobert, M. E., Karmali, V., Rao,

A. S., and Madara, J. L. (2000) Science 289(5484), 1560-3. 94. Melchior, F. (2000) Annu Rev Cell Dev Biol 16, 591-626 95. Orth, K., Palmer, L. E., Bao, Z. Q., Stewart, S., Rudolph, A. E., Bliska, J. B., and

Dixon, J. E. (1999) Science 285(5435), 1920-3. 96. Orth, K., Xu, Z., Mudgett, M. B., Bao, Z. Q., Palmer, L. E., Bliska, J. B., Mangel, W.

F., Staskawicz, B., and Dixon, J. E. (2000) Science 290(5496), 1594-7. 97. Spence, J., Sadis, S., Haas, A. L., and Finley, D. (1995) Mol Cell Biol 15(3), 1265-73. 98. Hofmann, R. M., and Pickart, C. M. (1999) Cell 96(5), 645-53. 99. Ulrich, H. D., and Jentsch, S. (2000) EMBO J 19(13), 3388-97. 100. Davies, K. J. (1995) Biochem Soc Symp 61, 1-31 101. Finkel, T., and Holbrook, N. J. (2000) Nature 408(6809), 239-47. 102. Rhee, S. G. (1999) Exp Mol Med 31(2), 53-9. 103. Salnikow, K., Su, W., Blagosklonny, M. V., and Costa, M. (2000) Cancer Res

60(13), 3375-8. 104. Li, N., and Karin, M. (1999) FASEB J 13(10), 1137-43. 105. Schreck, R., Rieber, P., and Baeuerle, P. A. (1991) EMBO J 10(8), 2247-58. 106. Schmidt, K. N., Amstad, P., Cerutti, P., and Baeuerle, P. A. (1995) Chem Biol

2(1), 13-22. 107. Bonizzi, G., Piette, J., Schoonbroodt, S., Greimers, R., Havard, L., Merville,

M. P., and Bours, V. (1999) Mol Cell Biol 19(3), 1950-60. 108. Flohe, L., Brigelius-Flohe, R., Saliou, C., Traber, M. G., and Packer, L. (1997)

Free Radic Biol Med 22(6), 1115-26 109. Israel, N., Gougerot-Pocidalo, M. A., Aillet, F., and Virelizier, J. L. (1992) J

Immunol 149(10), 3386-93. 110. Anderson, M. T., Staal, F. J., Gitler, C., and Herzenberg, L. A. (1994) Proc

Natl Acad Sci U S A 91(24), 11527-31. 111. Brennan, P., and O'Neill, L. A. (1995) Biochim Biophys Acta 1260(2), 167-75. 112. Suzuki, Y. J., Forman, H. J., and Sevanian, A. (1997) Free Radic Biol Med

22(1-2), 269-85 113. Suzuki, Y. J., and Packer, L. (1993) Biochem Mol Biol Int 31(4), 693-700. 114. Schoonbroodt, S., and Piette, J. (2000) Biochem Pharmacol 60(8), 1075-83. 115. Bonizzi, G., Piette, J., Merville, M. P., and Bours, V. (2000) Biochem

Pharmacol 59(1), 7-11. 116. Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J., and Hay, R. T.

(1992) Nucleic Acids Res 20(15), 3821-30.


117. Toledano, M. B., and Leonard, W. J. (1991) Proc Natl Acad Sci U S A 88(10), 4328-32.

118. Hayashi, T., Ueno, Y., and Okamoto, T. (1993) J Biol Chem 268(15), 11380-8. 119. Marshall, H. E., Merchant, K., and Stamler, J. S. (2000) FASEB J 14(13),

1889-900. 120. Chen, F., Kuhn, D. C., Sun, S. C., Gaydos, L. J., and Demers, L. M. (1995)

Biochem Biophys Res Commun 214(3), 839-46. 121. Bowie, A., and O'Neill, L. A. (2000) Biochem Pharmacol 59(1), 13-23. 122. Li, N., and Karin, M. (1998) Proc Natl Acad Sci U S A 95(22), 13012-7. 123. Korn, S. H., Wouters, E. F., Vos, N., and Janssen-Heininger, Y. M. (2001) J

Biol Chem 276(38), 35693-700. 124. Sanlioglu, S., Williams, C. M., Samavati, L., Butler, N. S., Wang, G., McCray,

P. B., Jr., Ritchie, T. C., Hunninghake, G. W., Zandi, E., and Engelhardt, J. F. (2001) J Biol Chem 276(32), 30188-98.

125. Jaspers, I., Zhang, W., Fraser, A., Samet, J. M., and Reed, W. (2001) Am J Respir Cell Mol Biol 24(6), 769-77.

126. Yin, Z., Ivanov, V. N., Habelhah, H., Tew, K., and Ronai, Z. (2000) Cancer Res 60(15), 4053-7.

127. Huang, R. P., Wu, J. X., Fan, Y., and Adamson, E. D. (1996) J Cell Biol 133(1), 211-20.

128. Kapahi, P., Takahashi, T., Natoli, G., Adams, S. R., Chen, Y., Tsien, R. Y., and Karin, M. (2000) J Biol Chem 275(46), 36062-6.

129. Chen, Y. R., Shrivastava, A., and Tan, T. H. (2001) Oncogene 20(3), 367-74. 130. Chen, K., Vita, J. A., Berk, B. C., and Keaney, J. F., Jr. (2001) J Biol Chem

276(19), 16045-50. 131. Mendelson, K. G., Contois, L. R., Tevosian, S. G., Davis, R. J., and Paulson,

K. E. (1996) Proc Natl Acad Sci U S A 93(23), 12908-13. 132. Spiegelman, V. S., Stavropoulos, P., Latres, E., Pagano, M., Ronai, Z., Slaga,

T. J., and Fuchs, S. Y. (2001) J Biol Chem 276(29), 27152-8. 133. Gotoh, Y., and Cooper, J. A. (1998) J Biol Chem 273(28), 17477-82. 134. Liu, H., Nishitoh, H., Ichijo, H., and Kyriakis, J. M. (2000) Mol Cell Biol

20(6), 2198-208. 135. Chen, F., Demers, L. M., Vallyathan, V., Ding, M., Lu, Y., Castranova, V., and

Shi, X. (1999) J Biol Chem 274(29), 20307-12. 136. Chen, C. Y., Del Gatto-Konczak, F., Wu, Z., and Karin, M. (1998) Science

280(5371), 1945-9. 137. Pages, G., Berra, E., Milanini, J., Levy, A. P., and Pouyssegur, J. (2000) J Biol

Chem 275(34), 26484-91. 138. Schmidt-Supprian, M., Bloch, W., Courtois, G., Addicks, K., Israel, A.,

Rajewsky, K., and Pasparakis, M. (2000) Mol Cell 5(6), 981-92. 139. Aradhya, S., Courtois, G., Rajkovic, A., Lewis, R. A., Levy, M., Israel, A., and

Nelson, D. L. (2001) Am J Hum Genet 68(3), 765-71. 140. Aradhya, S., and Nelson, D. L. (2001) Curr Opin Genet Dev 11(3), 300-6.

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Contributor(s)Written 12-2001 Fei Chen, Jacquelyn Bower, Laurence M. Demers, Xianglin Shi


CitationThis paper should be referenced as such : Chen F, Bower J, Demers LM, Shi X . Upstream Signal Transduction of NF-kB Activation. Atlas Genet Cytogenet Oncol Haematol. December 2001 .URL : http://AtlasGeneticsOncology.org/Deep/NFKBID20033.html

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